Spatiotemporally controlled single cell sonoporation

Abstract

This paper presents unique approaches to enable control and quantification of ultrasound-mediated cell membrane disruption, or sonoporation, at the single-cell level. Ultrasound excitation of microbubbles that were targeted to the plasma membrane of HEK-293 cells generated spatially and temporally controlled membrane disruption with high repeatability. Using whole-cell patch clamp recording combined with fluorescence microscopy, we obtained time-resolved measurements of single-cell sonoporation and quantified the size and resealing rate of pores. We measured the intracellular diffusion coefficient of cytoplasmic RNA/DNA from sonoporation-induced transport of an intercalating fluorescent dye into and within single cells. We achieved spatiotemporally controlled delivery with subcellular precision and calcium signaling in targeted cells by selective excitation of microbubbles. Finally, we utilized sonoporation to deliver calcein, a membrane-impermeant substrate of multidrug resistance protein-1 (MRP1), into HEK-MRP1 cells, which overexpress MRP1, and monitored the calcein efflux by MRP1. This approach made it possible to measure the efflux rate in individual cells and to compare it directly to the efflux rate in parental control cells that do not express MRP1.D espite the development of various approaches for transporting membrane-impermeant compounds (such as fluorescent markers, DNA, RNA, siRNA, proteins, peptides, and amino acids) into living cells (1-3), efficient intracellular delivery of bioactive agents for biomedical applications with minimal adverse effects remains challenging. In addition, it is desirable yet difficult to achieve local perturbation of intracellular processes, which requires subcellular molecular localization inside the living cell (4, 5).Sonoporation uses ultrasound to induce transient disruption of cell membranes (6-8), thereby enabling transport of membraneimpermeant compounds into the cytoplasm of living cells (6,(9)(10)(11). Without the need to use viral vectors, sonoporation enables the delivery of a wide range of bioactive agents with minimal inflammatory and immunological responses for both in vitro studies and in vivo applications (12-16). In addition, ultrasound application can be targeted to a specific volume of tissue in vivo noninvasively. These unique characteristics make sonoporation a compelling and versatile technology for nonviral drug and gene delivery.Sonoporation is typically performed for bulk treatment of a tissue volume in vivo or a large number of cells in vitro, often facilitated by microbubbles that are either injected in the vasculature or mixed in solution with suspended or attached cells. Ultrasound application induces cavitation of the microbubbles (17), signified by rapid volume expansion/contraction and/or collapse (18). These effects generate localized fluid flow, shear stress, and other mechanical or physical impact capable of affecting cells and structures nearby (7,19,20).However, the detailed processes supporting sonoporationmediated transmembrane and ...

Enhancement of Non-Invasive Trans-Membrane Drug Delivery Using Ultrasound and Microbubbles During Physiologically Relevant Flow

“…The low pressure and pulse length ensured safe and efficient drug delivery (figure 3(e) and table 1), confirming previous results indicating that intact and stably oscillating 15 microbubbles are beneficial for the sonoporation efficiency (Fan et al, 2012;van Wamel et al, 2006).…”

“…PI (P4170-100MG; Sigma-Aldrich, St. Louis, MO, USA) is a red fluorescent dye with an 10 excitation wavelength of 536 nm and an emission wavelength of 617 nm; fluorescent emission is triggered upon its binding with the double stranded nucleic acids. PI is normally impermeable to the cell membrane of viable cells despite its relatively small size (668.4 Da) and has been previously used as sonoporation indicator (Fan et al, 2012;Kooiman et al, 2011;Park et al, 2011;van Wamel et al, 2006). Successful trans-15 membrane delivery would thus facilitate the passage of PI into the nucleus of the cell while maintaining viability and would result in the co-localization of green and red fluorescence.…”

Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis

“…Moreover, when increasing the acoustic pressure, the distribution of cells shifted to the high uptake population. Indeed, acoustic pressure was shown to correlate with the size and the number of pores [10]. In conclusion, at low pressures endocytic uptake is promoted, while at higher pressure the fraction of cells in the pore formation population increases.…”

“…However, this technique does not provide information on drug uptake. Fluorescence microscopy can be used to monitor the uptake of fluorescent probes, serving as model drugs [10,39]. Besides using fluorescent probes, we fluorescently labeled microbubbles as well as cells.…”

“…Scanning electron microscopy images showed clear membrane disruptions after ultrasound exposure [9]. By voltage clamp techniques, an increase in transmembrane current simultaneously with ultrasound application was reported, indicating ion flux through pores [10]. Moreover, several papers demonstrated the uptake of cell-impermeable molecules when exposing cells to ultrasound [11][12][13].…”